Method and apparatus for performing contactless optically-induced dielectrophoresis for separation of circulating tumor cells

ABSTRACT

A method for performing contactless ODEP for separation of CTCs is provided with the steps of obtaining patients&#39; blood with rare cell suspected CTCs; adding at least one fluorescent antibody binding to CTCs into the blood; staining the blood; injecting the stained blood with fluorescent dye into an ODEP device and then performing fluorescent image identification; trapping the CTCs with at least one fluorescent antibody in the ODEP device by creating an image pattern and then generating an ODEP force; Separating the trapped CTCs from other non-CTCs cells; absorbing the trapped CTCs; and obtaining a high purity of CTCs. An apparatus for performing contactless ODEP for separation of CTCs is also provided.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention relates to methods and apparatuses for separation ofcirculating tumor cells (CTCs) and more particularly to a method andapparatus for performing contactless optically-induced dielectrophoresis(ODEP) for the separation of CTCs in which an initial cells separationis done using a conventional reagent separation technique, micro fluidand ODEP are employed, and images of fluorescent antibody are used toselect and separate the CTCs from the target cells. 100% purity of CTCscan be obtained for subsequent gene analysis.

2. Description of Related Art

Methods for the separation of CTCs from the blood are well known in theart. For example, Ficoll solution is used to separating red blood cells(RBCs) from the blood, magnetic beads of antibody are used to capturewhite blood cells (WBCs), micro fluid electrodes are used by a chemicalor physical method to separate blood cells from the blood, etc.

However, purity of the obtained CTCs is less than 100%. That means onlytens to hundreds CTCs are available for high precision sub-generationgene order analysis and they may be seriously interfered by other bloodcells. As a result, the gene order analysis is not correct andunacceptable.

Thus, the need for improvement still exists.

SUMMARY OF THE INVENTION

It is therefore one object of the invention to provide a method forperforming contactless optically-induced dielectrophoresis (ODEP) forseparation of circulating tumor cells (CTCs), the method comprising thesteps of obtaining patients' blood with rare cell suspected CTCs; addingat least one fluorescent antibody binding to CTCs into the blood;staining the blood; injecting the stained blood with fluorescent dyeinto an ODEP device and then performing fluorescent imageidentification; trapping the CTCs with at least one fluorescent antibodyin the ODEP device by creating an image pattern and then generating anODEP force; Separating the trapped CTCs from other non-CTCs cells;absorbing the trapped CTCs; and obtaining a high purity of CTCs.

It is another object of the invention to provide an apparatus forperforming contactless optically-induced dielectrophoresis forseparation of circulating tumor cells for the implementation of themethod, the apparatus comprising an ODEP device including a firstconductive glass, a bio-compatible membrane, and a second conductiveglass wherein the bio-compatible membrane is disposed below the firstconductive glass, the bio-compatible membrane includes a transverse mainchannel and a longitudinal micro channel perpendicular to the mainchannel and joining the main channel at a cell separation zone; thefirst conductive glass includes a first hole and a second hole alignedwith two ends of the main channel respectively, and a third hole alignedwith one end of the micro channel; and the second conductive glass isdisposed below the bio-compatible membrane; a sample receiving memberdisposed on and aligned with the first hole; an exhaust discharge memberdisposed on and aligned with the second hole; a target collection memberdisposed on and aligned with the third hole; and a controller includingan optical projection device and an image fetch device.

It is a further object of the invention to provide an apparatus forperforming contactless optically-induced dielectrophoresis forseparation of circulating tumor cells for the implementation of themethod, the apparatus comprising an ODEP device including a firstconductive glass and a second conductive glass wherein the firstconductive glass is disposed on the second conductive glass; the firstconductive glass includes a transverse main channel and a longitudinalmicro channel perpendicular to the main channel and joining the mainchannel at a cell separation zone; and the first conductive glassincludes a first hole and a second hole aligned with two ends of themain channel respectively, and a third hole aligned with one end of themicro channel; a sample receiving member disposed on and aligned withthe first hole; an exhaust discharge member disposed on and aligned withthe second hole; a target collection member disposed on and aligned withthe third hole; and a controller including an optical projection deviceand an image fetch device.

The invention has the following advantages and benefits in comparisonwith the conventional art: CTCs sample having a 100% purity is madepossible. Required fluorescent antibody of CTCs can be marked foranalysis as long as the fluorescent mark can be identified by an imagingsystem. Target cells can be obtained. White blood cells (WBCs) of acancer patient, cancer stem cells in the CTCs, and CTCs of high transferhaving surface antigens can be marked by the fluorescent antibody ofCTCs for medical search. A method for precise separation of CTCs is madepossible. The separated CTCs from a cancer patient can be used toreplace high risk samples obtained from cancer cells of a cancerpatient. Cancer cells gene analysis is made easy since effective samplescan be safely obtained from a cancer patient. Healed condition of acancer patient can be determined correctly. The high purity CTCs are notaffected by healthy cells and thus a next generation sequencing (NGS)can be performed on CTCs of a cancer patient. Medical effects ofanti-cancer medicine administered to a cancer patient can be wellobserved. Medicine companies can use the invention for new drugdevelopment. Precision and effectiveness of CTCs gene analysis aregreatly increased.

The above and other objects, features and advantages of the inventionwill become apparent from the following detailed description taken withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of a method for performing contactlessoptically-induced dielectrophoresis for separation of circulating tumorcells according to the invention;

FIG. 2 is a block diagram of an apparatus for performing contactlessoptically-induced dielectrophoresis for separation of circulating tumorcells according to the invention;

FIG. 3 is a perspective view of the ODEP device of a first preferredembodiment;

FIG. 4 is an exploded view of the ODEP device shown in FIG. 3;

FIG. 5 is an exploded view of an ODEP device of a second preferredembodiment;

FIG. 6 is an exploded view of an ODEP device of a third preferredembodiment; and

FIG. 7 illustrates scanning electron microscopy images of the separationof circulating tumor cells according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Optically-induced dielectrophoresis (ODEP) of the invention employslight as a virtual electrode. In detail, light impinges on light guidematerials of two tablet electrodes respectively. Uneven electric fieldsare generated around optical patterns. Polarized particles can bemanipulated by the uneven electric fields. In an operation, anindividual may operate a personal computer to direct light projectedfrom a projector to the particles for manipulation purposes.

Referring to FIG. 1, a method for performing contactlessoptically-induced dielectrophoresis (ODEP) for separation of circulatingtumor cells (CTCs) in accordance with the invention is illustrated. Themethod comprises the following steps as discussed in detail below.

Step 1: Obtaining blood with rare cell suspected CTCs.

Step 2: Adding at least one fluorescent antibody binding to CTCs intothe blood.

Step 3: Staining the blood.

Step 4: Injecting the stained blood with fluorescent dye into an ODEPdevice and then performing fluorescent image identification.

Step 5: Trapping the CTCs with at least one fluorescent antibody in theODEP device by creating an image pattern and then generating an ODEPforce.

Step 6: Separating the trapped CTCs from other non-CTCs cells.

Step 7: Absorbing the trapped CTCs.

Step 8: Obtaining a high purity of CTCs after repeating steps 5 and 6.

The red blood cells (RBCs) and the white blood cells (WBCs) can beremoved from the obtained blood between step 1 and step 2. Such RBCs andWBCs removal step can increase CTCs collection speed after blood beingstained. The removal of the RBCs can be done by a physical separationmethod or a chemical removal method. The removal of the WBCs can be doneby the group identification of CD45 cocktail magnetic antibody. Also,the WBCs having the expression of CD45 can be removed in cell nuclei.

Before the absorption of the trapped CTCs in step 7, the purity of thetrapped CTCs can be identified again by observing the fluorescentimages. The purpose of such identification is to determine whether thenon-target cells having fluorescent expression are there. If yes, thenon-target cells are identified by the fluorescent images in order todetermine whether they have cell nuclei and determine whether they arepseudo positive target cells. Thus It can further increase the purity ofthe trapped CTCs.

The physical method of removing RBCs from the blood comprises thefollowing steps of (a) adding a material capable of separating cellshaving a high density from cells having a low density to a test tube,and adding blood to the test tube to obtain a solution in whichvolumetric ratio of the material to the blood is 3:4; (b) rotating thetest tube at a centrifugal speed of 400 rpm for 30 minutes to convertthe solution in the test tube into an upper portion of serum, anintermediate portion of transparent product, and a lower portion ofRBCs; (c) removing blood having a single cell nucleus at a milky whiteinterface between the upper portion of serum and the intermediateportion of transparent product from the test tube and adding same toanother test tube; (d) using saline to wash the blood for apredetermined number of times; and (e) obtaining blood without RBCs.

Preferably, the material is either sugar or polysaccharide.

The chemical method of removing RBCs from the blood employs osmoses toexplode RBCs of the blood without cell nuclei in order to obtain WBCshaving cell nuclei. The chemical method comprises the following steps of(a) preparing a reagent 1×RBC lysis buffer 1,000 ml in a test tube bymixing 8.26 g of NH₄Cl, 1.19 g of NaHCO₃, 200 μL 0.5 M pH8 of EDTA, and1,000 ml of distilled water without microorganisms until the reagent haspH7.3; (b) adding blood to the reagent in which volumetric ratio of theblood to the reagent is 1:5, and reacting for 10 minutes; (c) rotatingthe test tube at a centrifugal speed of 400 rpm for 5 minutes to removethe upper portion of serum; (d) using 10 ml of PBS to wash the bloodcells once and rotating the test tube at a centrifugal speed of 400 rpmfor 5 minutes to remove the upper portion of serum; and (e) obtainingWBCs and CTCs having cell nuclei from the remains of the test tube.

In step 2, the fluorescent antibody is a unique antibody for binding tothe CTCs. The fluorescent antibody can collect and combine thosesuspected tumor cells having a weak CD45 expression or none CD45expression. The unique antibody for binding to the CTCs is comprised ofadhesion protein of epithelium, keratin protein of cells, surfaceantigens of cancer stem cell, epidermal growth factor receptor, andadhesion protein of calcium.

The high purity of CTCs are used to detect cancer and heal wounds aftersurgery. Further, the high purity CTCs are used to analyze gene, testresistance of medicine, and test medicine.

Referring to FIGS. 2 to 4, an apparatus for performing contactlessoptically-induced dielectrophoresis for separation of circulating tumorcells in accordance with a first preferred embodiment of the inventioncomprises an ODEP device 5, a first conductive glass 10, abio-compatible membrane 12, and a second conductive glass 14. Thebio-compatible membrane 12 is disposed below the first conductive glass10. A transverse main channel 18 is provided on the bio-compatiblemembrane 12. A longitudinal micro channel 20 is provided on thebio-compatible membrane 12, is perpendicular to the main channel 18, andjoins the main channel 18 at a cell separation zone 22 in theintermediate portion of the main channel 18. A first hole 24 and asecond hole 26 are formed through the first conductive glass 10 andaligned with two ends of the main channel 18 respectively. A third hole28 is formed through the first conductive glass 10 and aligned with oneend of the micro channel 20. A sample receiving member 30 is provided onand aligned with the first hole 24. An exhaust discharge member 32 isprovided on and aligned with the second hole 26. A target collectionmember 34 is provided on and aligned with the third hole 28. The secondconductive glass 14 is provided below the bio-compatible membrane 12. Acontroller 60 includes an optical projection device 62 and an imagefetch device 64.

A first electrode channel 161 is provided along an edge of the firstconductive glass 10 and corresponds to the main channel 18. A secondelectrode channel 162 is provided to be perpendicular to the firstelectrode channel 161 and joins the first electrode channel 161. Thesecond electrode channel 162 corresponds to the micro channel 20.

The second conductive glass 14 includes a light guide layer 141 formedon a bottom. The light guide layer 141 is distal the first conductiveglass 10. The light guide layer 14 is configured to guide light.

The ODEP device 5 includes a fluid driver 52 and a signal generationdevice 54 connected to the fluid driver 52.

Referring to FIGS. 2, 5 and 6, an apparatus for performing contactlessoptically-induced dielectrophoresis for separation of circulating tumorcells in accordance with a second preferred embodiment of the inventioncomprises an ODEP device 5, a first conductive glass 10, and a secondconductive glass 14. The first conductive glass 10 is provided on thesecond conductive glass 14. A transverse main channel 18 is provided onthe first conductive glass 14. A longitudinal micro channel 20 isprovided on the first conductive glass 14, is perpendicular to the mainchannel 18, and joins the main channel 18 at a cell separation zone 22in the intermediate portion of the main channel 18. A first hole 24 anda second hole 26 are formed through the first conductive glass 10 andaligned with two ends of the main channel 18 respectively. A third hole28 is formed through the first conductive glass 10 and aligned with oneend of the micro channel 20. A sample receiving member 30 is provided onand aligned with the first hole 24. An exhaust discharge member 32 isprovided on and aligned with the second hole 26. A target collectionmember 34 is provided on and aligned with the third hole 28. Acontroller 60 includes an optical projection device 62 and an imagefetch device 64.

A first electrode channel 161 is provided along an edge of the firstconductive glass 10 and corresponds to the main channel 18. A secondelectrode channel 162 is provided to be perpendicular to the firstelectrode channel 161 and joins the first electrode channel 161. Thesecond electrode channel 162 corresponds to the micro channel 20.

The second conductive glass 14 includes a light guide layer 141 formedon a bottom. The light guide layer 141 is distal the first conductiveglass 10.

The ODEP device 5 includes a fluid driver 52 and a signal generationdevice 54 connected to the fluid driver 52.

In operation, both the first and second electrode channels 161 and 162are positive and the second conductive glass 14 is negative if the firstconductive glass 10 is positive. To the contrary, the second conductiveglass 14 is positive if the first conductive glass 10 is negative.

Referring to FIG. 7, details of the flow chart of FIG. 1 are discussedbelow. Blood sample is obtained from a cancer patient. RBCs and WBCs areremoved from the blood sample using a conventional method. The remainingblood sample is dyed with commercially available fluorescent antibody.The dyed blood sample it added to a sample receiving member. Fluorescentimage identification is performed on the dyed blood sample to trackfluorescent target CTCs of the flowing blood cells suspension fluid. Inresponse to appearing of the fluorescent target CTCs on the cellseparation zone, the blood cells suspension fluid is stopped flowing.The blood cells suspension fluid begins to flow again after collectingthe CTCs. Target CTCs and non-target CTCs (e.g., WBCs) are identified.It is to determine the target CTCs have nuclei and they are not pseudopositive fluorescent target cells or doped target CTCs. ODEP isperformed to select target CTCs. ODEP is performed to remove thenon-target CTCs (e.g., WBCs) from the selected target CTCs and place itin a target collection member. The target CTCs are collected in thetarget collection member. ODEP is performed to collect the target CTCsin a next stage. Fluorescent images 1 to 12 are used to identify purityof the CTCs and determine whether the CTCs have nuclei not pseudopositive target cells. Finally, the CTCs are absorbed for subsequentanalysis.

The operation time is 20 minutes in which 2.5 ul/min flowing speed iscarried out in the first ten minutes and a stop is carried out in thenext ten minutes. Cell size is 30 ul. The number of cells is 10⁴.

The invention has the following advantages and benefits in comparisonwith the conventional art: CTCs sample having a 100% purity is madepossible. Required fluorescent antibody of CTCs can be marked foranalysis as long as the fluorescent mark can be identified by an imagingsystem. Target cells can be obtained. White blood cells (WBCs) of acancer patient, cancer stem cells in the CTCs, and CTCs of high transferhaving surface antigens can be marked by the fluorescent antibody ofCTCs for medical search. A method for precise separation of CTCs is madepossible. The separated CTCs from a cancer patient can be used toreplace high risk samples obtained from cancer cells of a cancerpatient. Cancer cells gene analysis is made easy since effective samplescan be safely obtained from a cancer patient. Healed condition of acancer patient can be determined correctly. The high purity CTCs are notaffected by healthy cells and thus a next generation sequencing (NGS)can be performed on CTCs of a cancer patient. Medical effects ofanti-cancer medicine administered to a cancer patient can be wellobserved. Medicine companies can use the invention for new drugdevelopment. Precision and effectiveness of CTCs gene analysis aregreatly increased.

While the invention has been described in terms of preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modifications within the spirit and scope of theappended claims.

What is claimed is:
 1. A method for performing contactlessoptically-induced dielectrophoresis (ODEP) for separation of circulatingtumor cells (CTCs), the method comprising the steps of: (1) obtainingblood with suspected CTCs; (2) adding at least one fluorescent antibodythat binds to the CTCs into the blood to form stained blood; (3)injecting the stained blood with the at least one fluorescent antibodyinto a sample receiving member of an ODEP device wherein the ODEP deviceincludes a first conductive glass and a second conductive glass, thefirst conductive glass includes a transverse main channel and alongitudinal micro channel joining the main channel at a cell separationzone, and the first conductive glass includes a first hole and a secondhole aligned with two ends of the main channel respectively, a thirdhole aligned with one end of the microchannel that is distal to the cellseparation zone, the sample receiving member disposed on and alignedwith the first hole, an exhaust discharge member disposed on and alignedwith the second hole, a target collection member disposed on and alignedwith the third hole, and a controller including an optical projectiondevice and an image fetch device; (4) performing fluorescent imageidentification by immunofluorescent microscopic observation using theoptical projection device and the image fetch device to identify anddistinguish the CTCs bound to the at least one fluorescent antibody frompseudo positive target cells that have fluorescent expression and lackcell nuclei; (5) trapping the identified CTCs bound to the at least onefluorescent antibody in the ODEP device by creating an image pattern byprojecting a light image and then generating an ODEP force; (6)separating the trapped CTCs from other non-CTCs at the cell separationzone; (7) collecting the trapped CTCs in the target collection member;and (8) obtaining the collected CTCs from the target collection member.2. The method of claim 1, wherein red blood cells (RBCs) and white bloodcells (WBCs) are removed from the blood between step (1) and step (2).